PROJECT SUMMARY Legionella pneumophila (Lp) is the agent of Legionnaires' disease, an increasingly common form of pneumonia. In the lungs, Lp grows primarily in macrophages, and cytokines from damaged host cells trigger severe inflammation. Iron is vital to Lp growth in extra- and intracellular niches and for the ability of Lp to cause disease. For >20 years, my lab has served a leadership role in the study of Lp iron acquisition. We previously showed that Lp secretes a siderophore when grown in a deferrated chemically-defined medium (CDM) and that this siderophore is required for Lp growth in lungs. Recently, we determined the structure of the molecule, finding it to be a polycarboxylate identical to rhizoferrin, a siderophore that had been first discovered in fungi. Besides demonstrating the ability of purified rhizoferrin to mediate Lp iron uptake, we also characterized the genes and proteins involved in its biosynthesis, export, import, and regulation. Interestingly, past studies using rhizoferrin from fungi showed that the siderophore is able to bind other metals, including Zn, Cu, and Mn, suggesting that the role of rhizoferrin in Lp pathogenesis may be multi-faceted. Thus, we will determine, for the first time, if rhizoferrin mediates the uptake of other metals and whether it also has the capacity to bind (deplete) metals in host cells and thereby trigger cytokine release from infected macrophages and epithelia. Intriguingly, LbtU, the outer membrane receptor for Lp rhizoferrin, differs in structure from known siderophore receptors, including not having a periplasmic N-terminal tail that binds energy-transducing TonB. This, coupled with the fact that Lp does not encode TonB or partners ExbB and ExbD, implies that LbtU is a new type of receptor, mediating uptake that is distinct from current paradigms. Consequently, we aim to identify the protein(s) that interacts with LbtU, including the Lp correlate of TonB-ExbBD, and thereby establish a new mechanism for siderophore transport across bacterial membranes. While studying rhizoferrin, we uncovered evidence that Lp secretes two additional siderophores, with one also being made during Lp growth in deferrated CDM and the other being produced upon growth in complex, buffered yeast extract broth that is only moderately-low in iron. The latter siderophore was dependent on FrgA, an iron-regulated, LbtA-like protein that we had previously shown is needed for Lp infection of macrophages. Thus, structural determination of these siderophores and further mutant analyses, as in a humanized mouse model of pneumonia, will lead to increased understanding of both Lp iron acquisition and pathogenesis. This effort also offers the chance for discovering a new type of siderophore, with the knowledge that ?new? siderophores have potential for clinical usage, such as the treatment of iron overload or generation of derivatives that act as carrier for antibiotics.